crispr cas9 double nickase plasmid mic25  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: SLP2 and MIC13 synergistically coordinate MICOS assembly and crista junction formation

doi: 10.1016/j.isci.2024.111467

Figure Lengend Snippet:

Article Snippet: CRISPR-Cas9 double nickase plasmid: MIC25 , Santa Cruz Biotechnology , sc-413621-NIC.

Techniques: Virus, Recombinant, Protease Inhibitor, In Situ, Cloning, Transfection, Mass Spectrometry, Knock-Out, Double Knockout, Stable Transfection, Expressing, Plasmid Preparation, Control, shRNA, CRISPR, Software

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A–D) Quantitative polymerase chain reaction (qPCR) analyzing the gene transcript fold changes of Opa-1 and MICOS across aging: (A) Opa1 transcripts, (B) Mitofilin transcripts, (C) Chchd3 transcript, and (D) Chchd6 transcripts. (E) Western Blot of OPA1, mitochondrial dynamic proteins, and MICOS protein expression. For all panels, error bars indicate SEM, and Mann–Whitney tests were used for statistical analysis. Each dot represents an individual qPCR run (n=4). Significance values indicate ***P ≤ 0.001 and ****P ≤ 0.0001. For all western blotting experiments, n = 4.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A-E) Individual knockout (KO) of Opa1 , Mitofilin , Chchd3 , and Chchd6 and representative transmission electron micrographs. (F–H) quantification upon KO state of each MICOS gene and Opa1 (n = 10 cells) was performed in 3-D reconstruction: (F) average single mitochondrion area, (G) average single mitochondrion perimeter, (H) average single mitochondrion circularity index, and (I) average single mitochondrion length across individual MICOS KO. (J) 4′,6-diamidino-2-phenylindole (DAPI) staining, MitoPY1 (5 uM, 45 min at 370 c magnification of 60x), and merge channels in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (K) 4′,6-diamidino-2-phenylindole (DAPI) staining, MitoBright Deep Red (10 uM, 30 min at 37 0 c), DCFDA (10 uM, 30 min at 37 0 c, magnification of 60x), and merge channels in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (L) Plate reader-based reactive oxygen species (ROS) quantification. (M) Microscopy-based ROS quantification of MitoPY1 orange, (N) DCFDA, and (O) MitoSox Deep Red. For all statistical tests, a one-way ANOVA statistical test was performed with Dunnett’s multiple comparisons test. For 3D microscopy, each dot represents a mitochondrion, with their number varied between control (n=81), Opa1 KO (n=153), Chchd3 KO (n=139), Chchd6 KO (n=180), and Mitofilin KO (n=156). Significance values indicate *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 and ****P ≤ 0.0001; ns, not significant.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Knock-Out, Transmission Assay, Staining, Control, Transfection, Microscopy

Journal: bioRxiv

Article Title: The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

doi: 10.1101/2024.06.09.598108

Figure Lengend Snippet: (A) Representative traces of mitochondrial calcium uptake in scramble-siRNA (control), MIC60-siRNA ( MITOFILIN KD), and CHCHD6-siRNA ( CHCHD6 KD) transfected permeabilized HEK293 cells. (B) Percentage of mCa2+ uptake rate calculated from (C) representative traces of mitochondrial calcium retention capacity in control, MITOFILIN KD, and CHCHD6 KD HEK293 cells. The number of boluses of calcium taken up by cells is shown in circles. (D) Percentage change in mitochondrial calcium retention capacity calculated from representative traces of mitochondrial calcium retention capacity. (E) Western blot showing siRNA-mediated KD of CHCHD6 /CHCHD6 in HEK293 cells. (F) Western blot showing siRNA-mediated KD of MITOFILIN /MIC60 in HEK293 cells. (G) Serial block face scanning electron microscopy obtained representative images of mitochondria endoplasmic reticulum contact site morphology overlaid on orthoslice and (H) isolated in three dimensions in three-month and (I-J) 2-year samples. For all statistical tests, one-way ANOVA statistical test was performed with Dunnett’s multiple comparisons test. N=3-5 for all calcium experiments, as run in triplicates. Significance values indicate **P ≤ 0.01.

Article Snippet: All cell types were infected with the following adenoviruses for gene knockouts: control CRISPR/Cas9 (sc-418922), CHCHD6 CRISPR (sc-425817), CHCHD3 CRISPR (sc-425804), and mitofilin CRISPR (sc-429376) (Santa Cruz Biotechnology, California, US), alongside appropriate guide RNAs ( ).

Techniques: Control, Transfection, Western Blot, Blocking Assay, Electron Microscopy, Isolation